Hemostatic compositions and methods for controlling bleeding

ABSTRACT

The invention provides hemostatic compositions useful to promote hemostasis at active bleeding wound sites. The hemostatic compositions typically include an article containing cellulose, e.g., cotton gauze, and a polysaccharide covalently linked to the cellulose, or a polysaccharide ionically cross-linked and in association with the article. Methods of making and using the hemostatic compositions are also provided.

CROSS REFERENCED TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/357,770 filed Feb. 17, 2006, which is a divisional of U.S. patentapplication Ser. No. 10/334,864 filed Dec. 31, 2002, which claimspriority from U.S. Provisional Patent Application No. 60/343,247, filedDec. 31, 2001, and from U.S. Provisional Patent Application No.60/354,917, filed Feb. 11, 2002, all of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This invention relates to hemostatic compositions and methods employingthe same, and more particularly to hemostatic compositions useful forcontrolling bleeding at active bleeding wound sites.

BACKGROUND

Wounds are generally classified as acute or chronic in accordance withtheir healing tendencies. Acute wounds, typically those received as aresult of surgery or trauma, usually heal uneventfully within anexpected time frame. Acute wounds include wounds such as active bleedingwound sites, e.g., wounds that have detectable, unclotted blood. Therapid control of topical bleeding at active bleeding wound sites is ofcritical importance in wound management, especially for the managementof trauma, e.g., as a result of military exercises or surgery.

A conventional method of controlling bleeding at active bleeding woundsites, such as an external hemorrhage or a surgical wound, advocates theuse of cotton gauze pads capable of absorbing 250 ml of blood. Cottonpads are considered passive, however, because of their inability toinitiate or accelerate blood clotting. Other formulations have beenreported to promote hemostasis and are described in U.S. Pat. Nos.6,454,787; 6,060,461; 5,196,190; 5,667,501; 4,793,336; 5,679,372;5,098,417; and 4,405,324. A hemostatic composition capable ofaccelerating the coagulation cascade to form a thrombus would be useful.

SUMMARY

Accordingly, the invention relates to hemostatic compositions andmethods for making and using the same in order to promote hemostasis atactive bleeding wound sites. The present compositions typically includean article which contains cellulose, e.g., cotton gauze, and apolysaccharide covalently linked to the cellulose. In other embodiments,a polysaccharide is ionically cross-linked and in association with anarticle comprising cellulose. Hemostatic compositions can includeadditional polysaccharides covalently linked to either or both of thecellulose and the first polysaccharide or physically trapped by anetwork formed by the covalent linking or ionic cross-linking of thefirst polysaccharide.

In one aspect of the invention, a method for controlling bleeding at anactive bleeding wound site of an animal is provided. The animal can be amammal. For example, the animal can be a human, horse, bird, dog, cat,sheep, cow, or monkey. The method includes applying a hemostaticcomposition to the active bleeding wound site. The hemostaticcomposition includes an article which contains cellulose and apolysaccharide, such as dextran, starch, or alginate, covalently linkedto the cellulose. If dextran is used, it may be in the form of a bead,e.g., covalently cross-linked dextran beads. The molecular weight of thedextran can range from about 10,000 to about 2,000,000 Daltons, or fromabout 20,000 to about 100,000 Daltons. When a polysaccharide is linkedto the cellulose, it can have a molecular weight exclusion limit ofgreater than about 30,000 Daltons.

Articles which contain cellulose can be barriers, structures, or devicesuseful in surgery, diagnostic procedures, or wound treatment. Forexample, an article containing cellulose can be a bandage, suture,dressing, gauze, gel, foam, web, film, tape, or patch. An articlecontaining cellulose can include a cotton material, e.g., cotton gauze.The article can also optionally include adhesives or polymericlaminating materials.

Hemostatic compositions of the present invention are useful foraccelerating blood clotting at an active bleeding wound site. Prior tothe application of a hemostatic composition, an active bleeding woundsite may be characterized in that it bleeds at a rate of from about 0.5ml/min to about 1000 ml/min. After application of a hemostaticcomposition, the active bleeding wound site may bleed at a rate of lessthan 0.03 ml/min. For example, the rate of less than 0.03 ml/min. may beachieved in from about 2 to about 20 minutes, and in certain embodimentsin less than about 5 minutes.

A hemostatic composition can comprise a second polysaccharide covalentlylinked to the cellulose and, optionally, to the first polysaccharide.The second polysaccharide may have a different molecular weight than thefirst polysaccharide. For example, the second polysaccharide may bedextran having a molecular weight from about 800,000 to about 2M.Alternatively, the second polysaccharide may be physically trapped bythe covalent linking of the first polysaccharide to the cellulose.

In other embodiments, hemostatic compositions of the present inventioncan include an article comprising cellulose in association with apolysaccharide ionically linked to itself (cross-linked). For example,the article comprising cellulose may be coated with, immersed in, orsoaked in the polysaccharide, which is subsequently ionicallycross-linked. The polysaccharide may be further covalently linked to thecellulose of the article. In addition, in certain embodiments, thepolysaccharide may be physically trapped in fibers of the articlecomprising cellulose.

One example of a polysaccharide that can be ionically cross-linked isalginate. Alginate can be ionically cross-linked to itself with metalcations, including Mg2+; Ni2+; Ca2+; Sr2+; Ba2+; Zn2+; Cd2+; Cu2+; Pb2+;Fe3+; and Al3+. In some embodiments, the cation is Ca2+. A secondpolysaccharide, such as dextran, can also be physically trapped, e.g.,by the network formed by the ionic cross-linking of the firstpolysaccharide. Dextran can be in the form of cross-linked beads, e.g.,dextran that has been previously cross-linked to itself. Dextran can becovalently linked to the bandage, e.g. by linking dextran to thecellulose with epichlorohydrin.

In another aspect, a hemostatic composition can include dextran-alginatespheres, such as ionically linked dextran-alginate spheres, orcovalently linked dextran-alginate spheres, or both ionically andcovalently linked dextran-alginate spheres.

In another aspect of the invention, hemostatic compositions are providedthat include additional agents, such as analgesics, steroids,antihistamines, anesthetics, bactericides, disinfectants, fungicides,vasoconstrictors, hemostatics, chemotherapeutic drugs, antibiotics,keratolytics, cauterizing agents, antiviral drugs, epidermal growthfactor, fibroblast growth factors, transforming growth factors,glycoproteins, collagen, fibrinogen, fibrin, humectants, preservatives,lymphokines, cytokines, odor controlling materials, vitamins, andclotting factors.

The invention also provides methods for making hemostatic compositions.Hemostatic compositions of the present invention can be made byincubating a linking agent with a polysaccharide and an articlecomprising cellulose to form a hemostatic composition having thepolysaccharide covalently linked to the cellulose.

The linking agent may be any linking agent useful for linking availablehydroxyl groups on cellulose with available hydroxyl groups on apolysaccharide. Examples include epichlorohydrin, dichlorohydrin,diepoxyburan, disepoxypropyl ether, orethylene-glyco-bis-epoxypropylether. The incubation step may occur in anaqueous alkaline solution. The temperature of the incubation step canrange from about 40° C. to about 70° C. In certain embodiments, thetemperature is about 50° C. The incubation step can occur for about 1 toabout 24 hours. In addition, the incubation step can be in the presenceof a stabilizing solution, e.g., a solution designed to prevent or limitevaporation of water. The stabilizing solution can include celluloseacetate butyrate. The covalently linked polysaccharide may have amolecular weight exclusion limit of greater than 30,000 Daltons.

In certain embodiments of the method, the polysaccharide is dextran. Thedextran can be in the form of covalently cross-linked beads. Themolecular weight of the dextran can range from about 10,000 to about 2M,or from about 20,000 to about 100,000 Daltons. The incubation step maybe occur in an aqueous alkaline solution having about 12% to about 75%dextran.

In another aspect, the invention provides a method of making acomposition including incubating a polysaccharide and a cation with anarticle containing cellulose in order to form a hemostatic compositionhaving the article containing cellulose in association with an ionicallycross-linked polysaccharide. The polysaccharide may be furthercovalently linked to the cellulose. The cation may be, for example,Ca2+. The Ca2+ may be in the form of, or derived from, Ca2+-loadedcross-linked dextran beads. The polysaccharide may be sodium alginate ora derivative of alginic acid, including salts of alginic acid.

In certain embodiments, the incubation step includes a secondpolysaccharide; the second polysaccharide may become physically trappedin the three dimensional network formed by the ionic cross-linking ofthe first polysaccharide. The second polysaccharide may be dextran,e.g., dextran in the form of cross-linked beads. The secondpolysaccharide may be further covalently linked to the cellulose, e.g.,through a linking agent such as epichlorohydrin.

In another aspect, the invention provides a method for manufacturing acomposition, where the method includes the step of mixing an aqueousphase alkaline polysaccharide solution with an organic phase stabilizingagent solution to form a mixture having polysaccharide spheres;incubating a cross-linking agent with the mixture to cross-link thepolysaccharide spheres; isolating the cross-linked polysaccharidespheres; and coating an article comprising a sodium alginate solutionwith the cross-linked polysaccharide spheres. The method can includeremoving the organic phase stabilizing agent from the mixture, e.g.,prior to isolating the cross-linked polysaccharide spheres. The methodcan also include exposing the cross-linked polysaccharide spheres to asolution comprising Ca2+ ions, e.g., washing the cross-linkedpolysaccharide spheres in a Ca2+ solution. The polysaccharide may bedextran, and the organic phase stabilizing agent solution may includecellulose acetate butyrate.

In certain embodiments of the method, cross-linked polysaccharidespheres are between about 30 to about 500 μm in size. The mixing andincubating steps may occur at a temperature of from about 40° C. toabout 70° C. The coating step can include spraying the article with thecross-linked polysaccharide spheres. The invention also relates tohemostatic compositions manufactured according to the above method.

In a further aspect, another method for manufacturing a composition isprovided. The method includes the steps of providing an aqueous phasealkaline solution having dextran and sodium alginate therein; preparingdextran-alginate spheres from the aqueous phase alkaline solution; andincubating the dextran-alginate spheres with a linking agent to linksaid dextran-alginate spheres. Dextran-alginate spheres can be preparedby any method conventional in the art, including the use of a mechanicaldroplet generator. A linking agent may covalently or ionically link orcross-link the dextran-alginate spheres. Accordingly, a linking agentmay be epichlorohydrin or a Ca2+-containing salt such as calciumchloride. The dextran-alginate spheres may be linked with a Ca2+ linkingagent, and then linked with an epichlorohydrin linking agent, or thelinking can be performed in the reverse order, or simultaneously. Themethod can further including coating, e.g., spraying, an article, suchas an article comprising sodium alginate, with the linkeddextran-alginate spheres. The invention also includes hemostaticcompositions manufactured according to the method.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patents, patent applications, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not meant to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

As used herein, the terms “linking” or “linked” are meant to indicateeither a covalent or ionic link, either direct or mediated by a chemicalmoiety or an ion, between two chemically distinct entities, e.g.,dextran linked to cellulose. The term “cross-link” is meant to indicatea covalent or ionic link, either direct or mediated by a chemical moietyor ion, between two chemically similar moieties, e.g., dextrancross-linked to itself, alginate cross-linked to itself. The chemicallysimilar moieties do not have to be identical. For example, dextranhaving a particular average molecular weight range includes dextranmolecules of a variety of molecular weights, and thus the dextranmolecules are not identical but chemically similar. When dextranmolecules having an average molecular weight range are linked, e.g.,covalently linked with epichlorohydrin, they are said to be“cross-linked.”

The terms “spheres,” “particles,” or “beads,” when used in the contextof the present invention, are not meant to imply different sizes, butare meant to be interchangeable terms describing an embodiment of acomposition.

The term “active bleeding wound site” means, at a minimum, thatunclotted blood is present in the wound, e.g., extravascular blood,particularly where the surface of a tissue has been broken or an artery,vein, or capillary system has been compromised. The rate of blood flowfrom an active bleeding wound site can vary, depending upon the natureof the wound. In some cases, an active bleeding wound site will exhibitblood flow at a rate from about at a rate of from about 0.5 ml/min toabout 1000 ml/min. Some active bleeding wound sites may exhibit higherrates of blood flow, e.g., punctures of major arteries such as theaorta. After application of the hemostatic composition, the activebleeding wound site may bleed at a rate of less than 0.03 ml/min. Forexample, the rate of less than 0.03 ml/min. may be achieved in fromabout 2 to about 20 minutes, and in certain embodiments in less thanabout 5 minutes.

Hemostatic Compositions

The invention relates to hemostatic compositions used to promotehemostasis at active bleeding wound sites. While not being bound by anytheory, it is believed that the hemostatic compositions of the presentinvention control bleeding by initiating and accelerating bloodclotting. The hemostatic compositions of the present invention activateplatelets and concentrate high molecular weight components of thecoagulation cascade (e.g., clotting factors) by excluding high molecularweight components of the cascade, while absorbing the lower molecularweight components in blood. Accordingly, coagulation cascade componentshaving a molecular weight higher than about 30,000 Daltons are excluded,including fibrinogen (MW 340,000); prothrombin (MW 70,000); thrombin (MW34,000); Factor V (MW 330,000); Factor VII (MW 50,000); Factor VIII (MW320,000); von Willebrand factor (MW>850,000); Factor IX (MW 57,000);Factor X (MW 59,000); Factor XI (MW 143,000); Factor XII (MW 76,000);Factor XIII (MW 320,000); high MW kininogen (Fitzgerald Factor) (MW120,000-200,000), and prekallikrein (Fletcher Factor) (MW85,000-100,000). In addition, laboratory experiments indicate thatplatelets aggregate around the hemostatic compositions of the presentinvention when exposed to blood. The net result is that concentratedclotting factors (coagulation cascade components) and activatedplatelets activate the conversion of prothrombin to thrombin in thepresence of Ca2+, which subsequently catalyzes the conversion offibrinogen to insoluble fibrin multimers, e.g., a fibrin clot.Additional information on the clotting cascade and hemostaticcompositions containing fibrin can be found in U.S. Pat. No. 5,773,033.

Hemostatic compositions typically include an article comprisingcellulose, e.g., cotton gauze, and a polysaccharide covalently linked tothe cellulose. In other embodiments, hemostatic compositions include anarticle comprising cellulose in association with a polysaccharide thatis ionically cross-linked. The polysaccharide can be further covalentlylinked to the cellulose. Hemostatic compositions can include additionalpolysaccharides covalently linked to either or both of the cellulose andthe first polysaccharide. Other embodiments of hemostatic compositionsinclude linked and cross-linked polysaccharide spheres, optionallyloaded with a cation, e.g., Ca2+.

It should be noted that certain hemostatic compositions comprise both amacroscopic structure (e.g., an article) and a microscopic structure(e.g., networks of polysaccharide cross-linkages or networks ofpolysaccharide covalent linkages to cellulose). Some hemostaticcompositions therefore form three dimensional networks of apolysaccharide, either as ionically linked chains or covalently bound tothe cellulose of the article. Accordingly, in some embodiments, a secondpolysaccharide may be physically trapped by the network formed by thefirst polysaccharide.

Accordingly, in one aspect, a hemostatic composition includes an articlecontaining cellulose and a polysaccharide, such as dextran, starch, oralginate, covalently linked to the cellulose. The article may includenatural or synthetic celluloses (e.g., cellulose acetate, cellulosebutyrate, cellulose propionate). The polysaccharide chosen should besafe for in vivo use, e.g., non-allergenic, non-toxic, and preferablynon-metabolized. Polysaccharides for clinical use are known in the artand available from a variety of sources. See, e.g., U.S. Pat. No.6,303,585.

As used herein, covalent linkages encompass bonds from any of theavailable chemical moieties of the polysaccharide to any of theavailable chemical moieties of the cellulose. For example, if thepolysaccharide dextran is used, hydroxyl moieties on dextran can becovalently linked to hydroxyl moieties on cellulose through the linkingagent epichlorohydrin. In that case, a glyceryl bridge linking dextranto cellulose is formed. For additional information, see Flodin, P., andIngelman, B., “Process for the Manufacture of Hydrophilic High MolecularWeight Substances,” British Patent No. 854,715; and Flodin, P. “Chapter2: The Preparation of Dextran Gels,” Dextran Gels and Their Applicationsin Gel Filtration, Pharmacia, Uppsala Sweden, 1962, pages 14-26.

The average molecular weight range of the polysaccharide can vary, buttypically ranges from about 10,000 to about 2M Daltons. The molecularweight range chosen will affect the molecular weight exclusion limit ofthe covalently linked polysaccharide, and thus its ability to excludethe coagulation components and concentrate them.

Dextran is a high molecular weight polysaccharide that is water-soluble.It is not metabolized by humans, and is non-toxic and tolerated well bymost animals, including humans. The average molecular weight of dextranused in the present invention can range from about 10,000 to about2,000,000 Daltons, or from about 20,000 to about 100,000 Daltons.

Dextran can be in the form of beads, e.g., covalently cross-linkedbeads, before it is linked covalently to the cellulose. Dextran beadscan exhibit a range of sizes, e.g., from about 30 to about 500 μm.Dextran beads are commercially available, e.g., as Sephadex™(Pharmacia); see, for example UK 974,054. Alternatively, dextran beadsor particles may be formed during the preparation of the hemostaticcomposition, e.g., from the covalent cross-linking of previouslyuncross-linked dextran molecules.

In other embodiments, dextran may be in solution form, e.g.,uncross-linked, before it is covalently linked to the cellulose. Dextranmay be covalently linked to the cellulose and covalently cross-linked toitself, e.g., when exposed to a linking agent such as epichlorohydrin.When dextran is in solution form (e.g., uncross-linked), the dextranmolecules may coat all or a component of the article, such as fibers ofa cotton bandage, so that it subsequently forms a three-dimensionalmicroscopic linked network or mesh when it is covalently linked to thecellulose and covalently cross-linked to itself. Dextran beads linked tocellulose or a cellulose-dextran mesh as described previously contributeto the ability of a hemostatic composition to exclude high molecularweight components of the coagulation cascade.

The average molecular weight of the polysaccharide, the degree oflinking of the polysaccharide to cellulose, and any cross-linking of thepolysaccharide (e.g., to itself) are factors in the molecular weightexclusion limit of the polysaccharide in a hemostatic composition andthe water regain of a hemostatic composition. Water regain is defined asthe weight of water taken up by 1 g of dry hemostatic composition andcan be determined by methods known in the art. For example, it is knownthat small changes in dextran concentration or linking agentconcentration (e.g., epichlorohydrin) can result in dramatic changes inwater regain. Typically, at lower molecular weights of dextran, a higherwater regain results. See Flodin, P., “Chapter 2: The Preparation ofDextran Gels,” Dextran Gels and Their Applications in Gel Filtration,Pharmacia, Uppsala Sweden, 1962, pages 14-26.

Similarly, the degree of hydration of the polysaccharide also affectsthe molecular weight exclusion limit. As the degree of hydrationincreases, the molecular weight exclusion limit of the polysaccharideusually increases. Typically, when dextran is linked to cellulose, thedextran will have a molecular weight exclusion limit of greater thanabout 30,000 Daltons, thus effectively excluding the components of thecoagulation cascade and concentrating them on the microscopic surface ofthe hemostatic composition.

Articles which contain cellulose can be any barriers, structures, ordevices useful in surgery, diagnostic procedures, or wound treatment.For example, an article containing cellulose can be a bandage, suture,dressing, gauze, gel, foam, web, film, tape, or patch. An articlecontaining cellulose can include a cotton material, e.g., cotton gauze.The article should allow the polysaccharide linked to the cellulose tointeract with the wound site.

A hemostatic composition can comprise a second polysaccharide covalentlylinked to cellulose. The second polysaccharide may have a differentmolecular weight than the first polysaccharide. For example, the secondpolysaccharide may be dextran having a molecular weight from about800,000 to about 2M. The second polysaccharide may be covalently linkedto the cellulose at a time after the first polysaccharide, at the sametime as the first polysaccharide, or at a time before the firstpolysaccharide.

In other embodiments, hemostatic compositions of the present inventioncan include an article containing cellulose in association with anionically cross-linked polysaccharide. The polysaccharide may be furthercovalently linked to the cellulose. In this context, ionic linkagesinclude ion-mediated bonds between available chemical moieties on thepolysaccharide. Typical chemical moieties that can be mediated with anion (e.g., a cation) include hydroxyl moieties. For example, sodiumalginate or alginic acid salts can be ionically linked with metalcations, including Mg2+, Ni2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Cu2+, Pb2+,Fe3+, and Al3+. Typically, Ca2+ may be used. The alginate can be of anytype, including type G (L-guluronic acid) or type M (D-mannuronic acid),or mixed M and G. For more information on alginate, see U.S. Pat. No.5,144,016.

In certain embodiments, a second polysaccharide, such as dextran, can bephysically trapped in the network formed by the ionic cross-linking ofthe first polysaccharide. Dextran can be in the form of covalentlycross-linked beads, e.g., dextran that has been previously cross-linkedto itself with epichlorohydrin, or Sephadex™ beads. Alternatively, thedextran can be in solution form (e.g., uncross-linked), as describedabove. In addition, dextran can be covalently linked to the cellulose,e.g. by linking dextran to the cellulose with epichlorohydrin.Accordingly, dextran may become cross-linked to itself.

Other embodiments of hemostatic compositions include dextran-alginatespheres, such as ionically linked dextran-alginate spheres, orcovalently linked dextran-alginate spheres, or both ionically andcovalently linked dextran-alginate spheres. In addition, cross-linkeddextran spheres loaded with Ca2+ ions are also included as hemostaticcompositions of the present invention.

Hemostatic compositions can include additional agents, such asanalgesics, steroids, antihistamines, anesthetics, bactericides,disinfectants, fungicides, vasoconstrictors, hemostatics,chemotherapeutic drugs, antibiotics, keratolytics, cauterizing agents,antiviral drugs, epidermal growth factor, fibroblast growth factors,transforming growth factors, glycoproteins, collagen, fibrinogen,fibrin, humectants, preservatives, lymphokines, cytokines, odorcontrolling materials, vitamins, and clotting factors. For furtherinformation on these additional agents for incorporation, refer to WO00/27327.

Hemostatic compositions may be used in combination with polymericlaminating materials and adhesives to provide both mechanical supportand flexibility to an article and to facilitate adhesion to the wound.Additional information on such polymeric laminating materials andadhesives for use in the present invention can be found in, e.g., WO00/27327.

Pharmaceutical Compositions

The present invention also contemplates pharmaceutical compositionscomprising certain hemostatic compositions of the present invention,e.g., dextran-alginate linked spheres or calcium-loaded cross-linkeddextran spheres. Pharmaceutical compositions may be formulated inconventional manners using one or more physiologically acceptablecarriers containing excipients and auxiliaries. Proper formulation isdependent upon the route of administration chosen.

A “pharmaceutically acceptable carrier” (also referred to herein as an“excipient”) is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or morehemostatic compositions to a subject. Pharmaceutically acceptablecarriers can be liquid or solid, and can be selected with the plannedmanner of administration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties, whencombined with a hemostatic composition. Other components may be presentin a pharmaceutical composition, if desired.

Pharmaceutical compositions of the present invention can be administeredby a number of methods depending upon the area to be treated.Administration can be, for example, topical or parenteral.Administration can be rapid (e.g., by injection) or can occur over aperiod of time. For treating tissues in the central nervous system,pharmaceutical compositions can be administered by injection or infusioninto the cerebrospinal fluid, preferably with one or more agents capableof promoting penetration of the pharmaceutical composition across theblood-brain barrier.

Formulations for topical administration include, for example, sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions in liquid or solid oil bases.Such solutions also can contain buffers, diluents and other suitableadditives. Pharmaceutical compositions and formulations for topicaladministration can include patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids, and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for parenteral administration can includesterile aqueous solutions, which also can contain buffers, diluents andother suitable additives (e.g., penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers).

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, aqueous suspensions, andliposome-containing formulations. These compositions can be generatedfrom a variety of components that include, for example, preformedliquids, self-emulsifying solids and self-emulsifying semisolids.Emulsions are often biphasic systems comprising of two immiscible liquidphases intimately mixed and dispersed with each other; in general,emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w)variety. Emulsion formulations have been widely used for oral deliveryof therapeutics due to their ease of formulation and efficacy ofsolubilization, absorption, and bioavailability.

Pharmaceutical compositions of the invention further encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. The term “pharmaceuticallyacceptable salts” refers to physiologically and pharmaceuticallyacceptable salts of the hemostatic compositions of the invention (i.e.,salts that retain the desired biological activity without impartingundesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); salts formed with organic acids (e.g., acetic acid, citricacid, oxalic acid, palmitic acid, or fumaric acid); and salts formedfrom elemental anions (e.g., chlorine, bromine, and iodine).

Methods of Controlling Bleeding

In one aspect of the invention, a method for controlling bleeding at anactive bleeding wound site of an animal is provided. The method includesapplying a hemostatic composition to the active bleeding wound site.Application of the hemostatic composition typically includes contactingthe hemostatic composition with the wound or bleeding site surface. Thehemostatic composition is maintained in contact with the wound orbleeding site for a period of time sufficient to control the bleeding,e.g., to clot the blood, slow the rate of bleeding, or stop thebleeding. The application may include the use of pressure, e.g., byusing an elastic bandage to maintain contact with the bleeding site.Alternatively, an internal wound may be packed with a hemostaticcomposition until hemostasis is achieved. In other embodiments, ahemostatic composition is delivered to the wound site. For example, acatheter or needle may be used to deliver a hemostatic composition to anintravascular puncture site or to a biopsy site. The catheter or theneedle may be optionally coated with a hemostatic composition of thepresent invention.

Usually a hemostatic composition can control bleeding, for example, to arate of less than 0.03 ml/min, in a period of from about 2 to about 20minutes. In certain embodiments, bleeding stops immediately, or in lessthan about 5 minutes.

Typically a hemostatic compositions of the present invention will beused to inhibit or completely stop bleeding of a parenchymal organ, suchas the liver, kidney, spleen, pancreas, or lungs; or to control bleedingduring surgery (e.g., abdominal, vascular, gynecological, dental, tissuetransplantation surgery, etc.). For example, percutaneous needlebiopsies are common interventional medical procedures. Possiblecomplications of needle biopsies, however, include bleeding at thebiopsy site. The amount of bleeding is related to the needle size,tissue sample size, location of the biopsy, and vascularization of thetissue. Hemostatic compositions of the present invention can be used topromote hemostasis at needle biopsy sites. Biopsy needles may either becoated with hemostatic compositions of the present invention, or may beused to deliver a hemostatic composition to the biopsy site. For moreinformation on biopsy tracts, see U.S. Pat. No. 6,447,534.

Similarly, catheterization and interventional procedures, such asangioplasty and stenting, generally are performed by inserting a hollowneedle through a patient's skin and muscle tissue into the vascularsystem. A guide wire is then typically passed through the needle lumeninto a blood vessel. The needle is removed and an introducer sheath isadvanced over the guide wire into the vessel, and a catheter istypically passed through the lumen of the introducer sheath and advancedover the guide wire for positioning. Upon completion of the medicalprocedure, the catheter and introducer sheath are removed, often leavinga puncture site in the vessel, with associated bleeding. Hemostaticcompositions of the present invention may be used to coat the exteriorof catheters, stents, introducer sheath, and guide wires, etc., or maybe delivered, e.g., via a catheter, to the puncture site in order topromote hemostasis. For additional information, see U.S. Pat. No.6,391,048.

The amount of hemostatic composition to be used will vary with thepatient, the wound, and the composition employed. For example,hemostatic compositions with varying water regains can be assembled(e.g., stacked in descending order) for use in major bleeding to attainhemostasis.

Methods for Making Hemostatic Compositions

In another aspect, the invention provides methods for making hemostaticcompositions. The hemostatic compositions of the present invention canbe made by incubating a linking agent with a polysaccharide and anarticle containing cellulose to form a hemostatic composition having thepolysaccharide covalently linked to the cellulose.

Any biologically compatible bifunctional or heterobifunctional reagentmay be used as the linking agent, including reagents with halogens,epoxides, hydroxy succinimide esters, aldehydes, activated thiols, orother moieties for reacting free amines, hydroxides, hydroxyls, orsulfhydryls on the bandage or on the polysaccharide. The bandage may bemodified, e.g., derivatized, to incorporate reactive moieties such asamines or sulfhydryls for reacting with a particular linking agent. Thepolysaccharide may also be modified, e.g., derivatized, in a similarmanner, provided that the polysaccharide so derivatized remainspharmaceutically suitable for animal, e.g., human use. The linking agentmay be epichlorohydrin, dichlorohydrin, diepoxyburan, disepoxypropylether, or ethylene-glyco-bis-epoxypropylether. For additionalinformation, see Flodin, P., and Ingelman, B., “Process for theManufacture of Hydrophilic High Molecular Weight Substances,” BritishPatent No. 854,715; and Flodin, P., “Chapter 2: The Preparation ofDextran Gels,” Dextran Gels and Their Applications in Gel Filtration,Pharmacia, Uppsala Sweden, 1962, pages 14-26.

The incubation step may occur in an aqueous alkaline solution.Typically, the polysaccharide is from about 10% to about 80% wt/vol ofthe aqueous alkaline solution. The concentration of the linking agent inthe incubation step can range from about 2% to about 20% wt/wt of thepolysaccharide.

The temperature of the incubation step can range from about 40° C. toabout 70° C. In certain embodiments, the temperature is about 50° C. Theincubation step can occur for about 1 to about 24 hours. The incubatingstep may also include agitation of the reagents. In addition, theincubation step can be in the presence of a stabilizing solution, e.g.,a solution designed to prevent or limit evaporation of water. Thestabilizing solution can include cellulose acetate butyrate. The methodcan also include neutralizing the aqueous alkaline solution, e.g., withan acid such as HCl at a concentration of from 1 to 5M.

The hemostatic composition can be washed with an aqueous solution, e.g.,distilled water, or an aqueous alcoholic wash, e.g., 50/50 vol/volEtOH/water. The hemostatic composition can be serially washed inincreasing amounts of an alcoholic wash, such as 25%, 50%, 75%, and 100%EtOH. The alcohol wash solution can contain a humectant, e.g., glycerin,at a concentration of about 0.1 to about 2.0%. The hemostaticcomposition can be dried, e.g., at about 50° C. to about 80° C. Forexample, the hemostatic composition can be dried at 70° C. After drying,the covalently linked polysaccharide may have a molecular weightexclusion limit of greater than 30,000 Daltons.

In certain embodiments of the method, the polysaccharide is dextran. Thedextran can be in the form of covalently cross-linked beads. Themolecular weight of the dextran can range from about 10,000 to about 2M,or from about 20,000 to about 100,000 Daltons. Typically, dextran of MW40,000 is used. The incubation step may be occur in an aqueous alkalinesolution having about 12 to about 75% dextran wt/vol.

In another aspect, the invention provides a method of making ahemostatic composition including incubating a polysaccharide and acation with an article containing cellulose in order to form ahemostatic composition having the article containing cellulose inassociation with an ionically cross-linked polysaccharide. Thepolysaccharide may be further covalently linked to the cellulose. Thecation may be as described previously, including, for example, Ca2+. TheCa2+ may be in the form of, or derived from, Ca2+-loaded cross-linked,dextran beads. The polysaccharide may be sodium alginate or derivativesof alginic acid, including salts of alginic acid. Aqueous and alcoholicwashes of the hemostatic composition can be performed, as describedpreviously.

In certain embodiments, the incubation step includes a secondpolysaccharide. The second polysaccharide may be dextran, e.g., dextranin the form of covalently cross-linked beads. The second polysaccharidemay be physically trapped, e.g., in the three-dimensional network formedby the ionic cross-linking of the first polysaccharide. The secondpolysaccharide may be further covalently linked to the bandage, e.g.,through a linking agent such as epichlorohydrin.

In one embodiment, an article such as cellulose gauze is immersed in asolution of a first polysaccharide (e.g., about 1 to 5% sodium alginate)and a second polysaccharide (e.g., 20% dextran, avg. molecular weight40,000). The first polysaccharide is ionically cross-linked with acation solution, e.g., Ca2+ from a solution having about 0.5 to about10% aqueous calcium chloride. The Ca2+ concentration can be reduced withserial washes, e.g., to reduce the Ca2+ concentration to about 0.5%Ca2+. The first or second polysaccharide, or both, may then becovalently linked to the cellulose and/or cross-linked using an aqueousalkaline solution (e.g., 20% NaOH) of a linking agent, e.g.,epichlorohydrin (e.g., at about 3-6% of the weight of the secondpolysaccharide). The resulting hemostatic composition may be dried asdescribed previously.

In another embodiment of the present invention, cross-linkedpolysaccharide spheres can be mixed with a Ca2+-alcoholic wash solution(e.g., 1% calcium chloride in neat alcohol). The cross-linkedpolysaccharide spheres can be purchased, e.g., as Sephadex™, or can beprepared from an aqueous alkaline polysaccharide solution and across-linking agent (e.g., dextran cross-linked with epichlorohydrin),as discussed previously. After washing in the Ca2+-alcohol solution, thecross-linked polysaccharide spheres have Ca2+ in their pores, e.g., areCa2+-loaded spheres, and can be used to coat an article, e.g., sprayedonto a bandage, that has been previously soaked or immersed in apolysaccharide solution, e.g., sodium alginate, at a concentration ofabout 0.5-5% polysaccharide. In certain embodiments, Ca2+ from thecross-linked spheres exchanges with the sodium from the sodium alginatesolution, resulting in ionically cross-linked calcium alginate, whichphysically traps cross-linked polysaccharide spheres in thethree-dimensional network of ionic bonding. The hemostatic compositionso formed may be dried as discussed previously.

In another aspect, the invention provides a method for manufacturing acomposition, where the method includes the step of mixing an aqueousphase alkaline polysaccharide solution with an organic phase stabilizingagent solution to form a mixture having polysaccharide spheres;incubating a cross-linking agent with the mixture to cross-link thepolysaccharide spheres; isolating the cross-linked polysaccharidespheres; and coating an article comprising a sodium alginate solutionwith the cross-linked polysaccharide spheres. The method can includeremoving the organic phase stabilizing agent from the mixture, e.g.,prior to isolating the cross-linked polysaccharide spheres. The methodcan also include exposing the cross-linked polysaccharide spheres to asolution comprising Ca2+ ions, e.g., washing the cross-linkedpolysaccharide spheres in a Ca2+ solution. The polysaccharide may bedextran, and the organic phase stabilizing agent solution may includecellulose acetate butyrate.

In certain embodiments of the method, the cross-linked polysaccharidespheres are between about 30 to about 500 μm in size. The mixing andincubating steps may occur at a temperature of from about 40° C. toabout 70° C. The coating step can include spraying the article with thecross-linked polysaccharide spheres. The invention also relates tohemostatic compositions manufactured according to the above method.

In a further aspect, another method for manufacturing a composition isprovided. The method includes the steps of providing an aqueous phasealkaline solution having dextran and sodium alginate therein; preparingdextran-alginate spheres from the aqueous phase alkaline solution; andincubating the dextran-alginate spheres with a linking agent to formlinked dextran-alginate spheres. The dextran-alginate spheres can beprepared by any method conventional in the art, including the use of amechanical droplet generator or an air knife.

The linking agent may covalently or ionically link and/or cross-link thedextran-alginate spheres. Accordingly, the linking agent may beepichlorohydrin or a cation as described previously, e.g., aCa2+-containing salt. The dextran-alginate spheres may be first linkedwith a cation linking agent, and then linked with an epichlorohydrinlinking agent, or vice versa. The dextran-alginate spheres may be linkedsimultaneously with a cation linking agent and an epichlorohydrinlinking agent. The method can further including coating, e.g., spraying,an article with the linked dextran-alginate spheres. The invention alsois directed to hemostatic compositions manufactured according to themethod.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1 Cotton-Dextran Compositions

Pharmaceutically acceptable cotton-based compositions were incubatedwith dextran (40,000 MW) in an alkaline epichlorohydrin solution (20%dextran in NaOH wt/vol; epichlorohydrin at about 3 to 6% wt/wt dextran).The solutions were allowed to react for about 1 to about 16 hours at atemperature range from about ambient room temperature to about 60° C.The resulting cross-linking reactions were subsequently neutralizedusing a 1 to 5 Molar HCl solution.

The cross-linked and linked dextran-cotton hemostatic compositions werewashed about 4 times with distilled water. The products were furtherwashed twice with a 50% distilled water/alcohol solution, then with a75% alcohol solution, and lastly with a 100% alcohol solution, to removeexcess epichlorohydrin. A final alcohol wash solution contained about0.1 to about 2% glycerin to keep the composition from becoming brittle.The hemostatic composition was dried at about 70° C. overnight.

Example 2 Cotton-Alginate-Dextran Compositions

A stabilizing agent, such as cellulose acetate butyrate was dissolved inethylene dichloride at 3% wt/vol and heated to about 50° C. whilestirring at about 200 RPM in a 1-2 liter cylindrical reaction vessel.Dextran (MW 40,000) was dissolved in water at 15% wt/vol with 5N NaOH at2% of the dextran weight. The dextran solution was gradually added tothe stabilizing mixture with continued heating and stirring. Whendroplets of the desired size were formed (30-500 μm), a cross-linkingagent such as epichlorohydrin was added to the vessel at 20% of thedextran weight. The reaction formed cross-linked gel spheres in 1-3hours, but was allowed to proceed up to 16 hours before termination.Acetone was added and decanted twice to remove the stabilizer (celluloseacetate butyrate). The spheres were then treated with NaOH (equal partsof 2N NaOH and 95% ethyl alcohol) for about 15 mins., and neutralizedwith dilute acid (1N HCl) before filtration and washing with water. Theswollen spheres were shrunk with alcohol treatment (25, 50, 75, 100%serial alcohol washes).

Dry calcium chloride was mixed with a second 100% alcohol wash solution(1% calcium chloride in alcohol), which was used to wash thecross-linked dextran particles. The alcohol was evaporated off, trappingthe calcium in the pores of the dextran particles. The final productswere dry cross-linked dextran-calcium ion compositions.

Pharmaceutically acceptable cotton-based compositions were immersed(dipped) into a sodium alginate liquid solution (0.5-4%). After removalfrom the solution, the wet sodium alginate coated cotton materials weresprayed or dusted with the cross-linked dextran calcium compositions.Calcium exchanged with sodium, resulting in cross-linked calciumalginate. The cotton-dextran-alginate hemostatic compositions were thendried at 70° C. overnight.

Example 3 Alternative Method to Prepare Cotton-Dextran-AlginateCompositions

Cotton gauzes were immersed in solutions of 1 to 5% sodium alginate and20% dextran (40,000 MW). The mixtures were cross-linked and linked withabout 0.5% to about 10% aqueous calcium chloride solution. Thecompositions were washed to reduce Ca2+ concentration to about 0.5%Ca2+.

Dextran-alginate cross-linking solutions were prepared using an aqueousalkaline epichlorohydrin solution, where the concentration of theepichlorohydrin was from about 3 to about 6% by weight of the dextran.The solutions included about 20% NaOH. The resulting hemostaticcompositions were allowed to dry at ambient to about 60° C. overnight.

Example 4 Cotton-Dextran Compositions

Dextran (MW 40,000) was dissolved in 1N NaOH at a range of 18-69% wt/volof dextran in the alkaline solution. Epichlorohydrin was added to aconcentration of 20% of the dextran by weight at room temperature.Pharmaceutically acceptable cotton (cellulose) gauzes were added to thealkaline epichlorohydrin solutions. The solutions were allowed to reactwith agitation at 25° C. to 70° C. The mixtures were heated until across-linked dextran-cellulose gel formed on the gauze fibers, from 1-6hours, up to 24 hours. After neutralization with dilute HCl (1N),successive washes removed excess reaction products and impurities: fourtimes in distilled water, then with increasing concentrations of alcohol(25, 50, 75, 100%). A final alcohol wash of 100% EtOH contained from 0.1to 2.0% glycerin to keep the compositions pliable. The resultinghemostatic compositions were dried at about 70° C. overnight.

The water regain for the hemostatic compositions ranged from 2.5 ml/g to35 ml/g. For information on water regain, see Flodin, P., Dextran Gelsand Their Applications in Gel Filtration, Pharmacia, Uppsala Sweden,1962, pages 31-32.

Example 5 Cellulose-Dextran Composition

150 g of dextran (MW 40,000) was dissolved in 300 ml 1N NaOH. 30 gepichlorohydrin was quickly mixed with the dextran solution at roomtemperature. A pharmaceutically acceptable cellulose gauze was dipped inthe alkaline epichlorohydrin solution to thoroughly coat the fibers withthe reaction solution, then placed in a flat-bottomed dish. The gauzewas heated to 50° C. in a humidified atmosphere, with gentle rockingafter 1 hour, until a dextran-cellulose gel formed on the gauze fibers,typically in 1-2 hours. Heating was continued until the desiredend-point, up to 24 hours. The gauze was neutralized, washed, treatedwith glycerin, and dried as described previously. The water regain was7.5 ml/g.

Example 6 Cellulose-Dextran Composition

Dextran (MW 40,000) was dissolved in 1N NaOH (34% dextran wt/vol of thealkaline solution). Epichlorohydrin was added to a concentration of 20%of the dextran by weight at room temperature. A pharmaceuticallyacceptable cellulose fiber based composition, 16-ply 4×4 gauze, wasdipped in the alkaline epichlorohydrin solution to thoroughly coat thefibers with the reaction solution, then placed in a flat bottomed dish.To prevent concentrating the reaction solution by evaporation, astabilizing solution of cellulose acetate butyrate in ethylenedichloride (3% wt/vol), which is immiscible in water, was used to coverthe gauze. The gauze was heated at 50° C., with gentle rocking after 1hr., until a cross-linked and linked dextran-cellulose gel formed on thegauze fibers, typically in about 2 to 3 hours. Heating was continueduntil the desired endpoint, up to 24 hours. Acetone was added anddecanted twice to remove the stabilizer. The gauze was neutralized withdilute HCl and washed as described previously in aqueous alcohol andalcohol solutions and dried at about 70° C. overnight. The water regainwas 15 ml/g.

Example 7 Cellulose Dextran Composition

Dextran (MW 40,000) was dissolved in 1N NaOH and reacted with gauze inthe presence of epichlorohydrin, followed by neutralization, washing,and drying as described above. Cellulose-dextran compositions were madeby varying the volume of dextran in the solvent from 12 to 75 wt/vol toproduce hemostatic composition with water regains ranging from 5 ml/g to35 ml/g. The compositions can be assembled for use in major bleeding bystacking them in descending order (e.g., 35 ml/g to 5 ml/g) to attainhemostasis.

Example 8 Dextran-Calcium Spheres

A non-ionic polymer substance was dissolved in a suitable solvent withan alkaline solution added as a cross-linking catalyst. A stabilizer wasdissolved in a solvent that was immiscible with the polymer solvent andplaced in a cylindrical vessel. The stabilizer solution formed thecontinuous phase and was heated with regular stirring. When the polymersolution was added to the stabilizer solution, a biphasic system formedin which the polymer droplets became the dispersed phase. A bifunctionalcross-linking agent was added to the system which causedco-polymerization (cross-linking) of the polymer to form gel spheres.

After purification and drying, the water regain of the spheres wasdetermined in order to classify the molecular sieving capability of thecross-linked polymer. Water regain can be determined by methods wellknown in the art, and generally involves hydrating 1 gram of drycomposition, and determining the amount of water absorbed by the 1 g ofthe dry composition. Generally, greater swelling capacity relates tolarger pores (e.g., less cross-linking) and a higher molecular weightexclusion.

More specifically, a stabilizing agent, such as cellulose acetatebutyrate was dissolved in ethylene dichloride at 3% wt/vol and heated toabout 50° C. while stirring at about 200 RPM in a 1-2 liter cylindricalreaction vessel. Dextran (MW 40,000) was dissolved in water at 15%wt/vol with 5N NaOH at 2% of the dextran weight. The dextran solutionwas gradually added to the stabilizing mixture with continued heatingand stirring. When droplets of the desired size were formed (30-500 μm),a cross-linking agent such as epichlorohydrin was added to the vessel at20% of the dextran weight. The reaction formed cross-linked gel spheresin 1-3 hours, but was allowed to proceed up to 16 hours beforetermination. Acetone was added and decanted twice to remove thestabilizer (cellulose acetate butyrate). The spheres were then treatedwith NaOH (equal parts of 2N NaOH and 95% ethyl alcohol) for about 15mins., and neutralized with dilute acid (1N HCl) before filtration andwashing with water. The swollen spheres were shrunk with alcoholtreatment (25, 50, 75, 100% serial alcohol washes) as describedpreviously. A final alcohol wash contained calcium chloride (0.04-1%).The alcohol was evaporated by drying at about 70° C., thus trapping thecalcium in the pores and on the surface of the dextran spheres. Thefinal product was a dry cross-linked dextran-calcium ion composition.The water regain was 20 ml/g.

Example 9 Cotton-Alginate-Dextran Compositions

A pharmaceutically acceptable cotton (cellulose) gauze was immersed inor sprayed with a sodium alginate solution (0.5-4%). The wet sodiumalginate coated gauzes were sprayed or dusted with dextran-calciumspheres, prepared as described previously. Calcium exchanged with sodiumin the alginate, resulting in cross-linked calcium alginate. Ionic bondsformed between cellulose, alginate, and dextran, thereby chemicallyincorporating the spheres into the gauze to form the hemostaticcompositions.

Example 10 Dextran-Alginate Spheres

43 g dextran (MW 40,000) was dissolved in 50 ml 2N NaOH and mixed with a50 ml solution of 2% sodium alginate (43% dextran and 1% alginate in 100ml of 1N NaOH). 50-200 μm dextran-alginate droplets were linked andcross-linked in a solution of 1.7% calcium chloride. The spheres werefurther linked and cross-linked by adding 7.2 ml epichlorohydrin to 100ml of the calcium chloride solution at 45° C. with agitation. Thereaction continued for up to 16 hours until the desired amount oflinking and cross-linking occurred. The spheres were neutralized withdilute HCl, washed in increasing concentrations of alcohol (25, 50, 75,100%) and dried at about 70° C. overnight. The water regain was 10 ml/g.

Example 11 Cotton-Dextran-Alginate Sphere Composition

Cross-linked and linked dextran-alginate spheres, prepared as describedpreviously, were washed in a final calcium chloride alcohol wash(0.04-1% calcium chloride). The alcohol was evaporated to trap thecalcium in the pores of the spheres. The spheres were then chemicallyincorporated into gauze containing sodium alginate, as describedpreviously.

Example 12 Compositions having Dextrans of Different Molecular Weights

100 g of dextran (MW 2,000,000) dissolved in 2000 ml 0.5N NaOH was mixedwith 30 g of dextran spheres, prepared as described previously. Thecross-linked spheres and dextran solutions were mixed, and 100 gepichlorohydrin added. A pharmaceutically acceptable cellulose gauze wasplaced in a flat bottomed dish and the alkaline epichlorohydrin/dextransphere/dextran solution was slowly poured over the gauze to thoroughlycoat the fibers with the dextran spheres and dextran solution The coatedgauze was heated at 50° C. with gentle rocking, until a gel formed onthe gauze fibers, typically in 1-2 hours. The coated gauze was removedfrom the solution and placed in a flat bottom dish and heating wascontinued until the desired end-point, approximately 8-12 hours. Thegauze was neutralized, washed, and dried, as described previously.

Example 13 Tests of Cotton-Alginate-Dextran Compositions andCalcium-Dextran Spheres

Two different alginates (G, M) were applied in 0.5% and/or 1%concentrations to 12-ply 2×2 cotton gauze squares, Type VII, in either 2or 4 mL amounts. 0.5 g of calcium-dextran spheres (containing 3, 6, 12,or 24 mM calcium) were ionically bonded to the sodium alginate on thegauze. Freshly drawn human blood was added to each test sample, with 0.5mls added to each calcium-dextran-alginate gauze and 0.1 ml added to thecalcium-dextran spheres alone. Gauze immersed only in sodium alginatewas used as a control.

The blood added to the calcium-dextran spheres clotted upon contact. Allof the calcium-dextran-alginate gauzes clotted the blood in less than 5minutes, whereas alginate only gauzes produced weakly clotted blood in>5mins. See Table 1 below. TABLE 1 Clotting Times for HemostaticCompositions gauze + 4 gauze + 4 gauze + 2 gauze + 4 ml .5% ml 1% ml 1%ml 1% alginate G alginate G alginate G alginate M 0 mM Ca++ >5 min. >5min. >5 min. >5 min. control 3 mM Ca++ instantly not tested <5 min. nottested not tested dextran spheres 6 mM Ca++ instantly <5 min. <5 min. <5min. <5 min. dextran spheres 12 mM instantly <5 min. <5 min. <5 min. <5min. Ca++ dextran spheres 24 mM instantly <5 min. <5 min. <5 min. <5min. Ca++ dextran spheres

1. A method for controlling bleeding at an active bleeding wound site ofan mammal, said method comprising applying a hemostatic composition tosaid active bleeding wound site, said hemostatic composition comprisingan article comprising cellulose and a polysaccharide covalently linkedto said cellulose.
 2. The method of claim 1, wherein said polysaccharideis selected from the group consisting of cross-linked dextran,cross-linked starch, and cross-linked alginate.
 3. The method of claim2, wherein said polysaccharide is cross-linked dextran.
 4. The method ofclaim 2, wherein the molecular weight of said cross-linked dextran isfrom about 10,000 to about 2,000,000.
 5. The method of claim 4, whereinthe molecular weight of said cross-linked dextran ranges from about20,000 to about 100,000.
 6. The method of claim 1, wherein saidcovalently linked polysaccharide has a molecular weight exclusion limitgreater than about 30,000 Daltons.
 7. The method of claim 1, whereinsaid article comprising cellulose is selected from the group consistingof a bandage, suture, dressing, gauze, gel, foam, web, film, tape, orpatch.
 8. The method of claim 1, wherein said article comprisingcellulose comprises cotton.
 9. The method of claim 8, wherein saidarticle comprising cellulose is cotton gauze.
 10. The method of claim 1,wherein prior to said application of said hemostatic composition, saidactive bleeding wound site bleeds at a rate from about 0.5 ml/min toabout 1000 ml/min.
 11. The method of claim 1, wherein after applicationof said hemostatic composition, said active bleeding wound site bleedsat a rate of less than 0.03 ml/min.
 12. The method of claim 11, whereinsaid rate of less than 0.03 ml/min is achieved from about 2 to about 20minutes after application of said hemostatic composition.
 13. The methodof claim 1, wherein said hemostatic composition further comprises asecond polysaccharide covalently linked to said cellulose.
 14. Themethod of claim 5, wherein said hemostatic composition further comprisesa second polysaccharide, wherein said second polysaccharide iscross-linked dextran having a molecular weight from about 800,000 toabout 2M, wherein said second polysaccharide is covalently linked tosaid cellulose.
 15. A method for controlling bleeding at an activebleeding wound site of an animal, said method comprising applying ahemostatic composition to said active bleeding wound site, saidhemostatic composition comprising an article comprising cellulose inassociation with ionically cross-linked alginate.
 16. The methodaccording to claim 15, wherein said alginate is ionically cross-linkedwith a cation selected from the group consisting of: Mg2+; Ni2+; Ca2+;Sr2+; Ba2+; Zn2+; Cd2+; Cu2+; Pb2+; Fe3+; and Al3+.
 17. The methodaccording to claim 15, further comprising a second polysaccharidecovalently linked to said cellulose, wherein said second polysaccharideis cross-linked dextran.
 18. The method according to claim 17, whereinsaid alginate is further covalently linked to said cross-linked dextran.19. The method of claim 1, wherein said mammal is a human.
 20. Themethod of claim 3, wherein said cross-linked dextran is in the form ofbeads.